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Characterization of the promoter and extended C-terminal domain of Arabidopsis WRKY33 and functional analysis of tomato WRKY33 homologues in plant stress responses.

Zhou J, Wang J, Zheng Z, Fan B, Yu JQ, Chen Z - J. Exp. Bot. (2015)

Bottom Line: We compared AtWRKY33 with its close homologues to identify AtWRKY33-specific regulatory and structural elements, which were then functionally analysed through complementation.Thus, WRKY33 proteins are evolutionarily conserved with a critical role in broad plant stress responses.Both its CTD and promoter are critical for the uniquely important roles of WRKY33 in plant stress responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China Department of Botany and Plant Pathology, 915W. State Street, Purdue University, West Lafayette, IN 47907-2054, USA jie@zju.edu.cn zhixiang@purdue.edu.

No MeSH data available.


Related in: MedlinePlus

Complementation of atwrky33 mutant plants for Botrytis resistance by AtWRKY33 (AtW33), AtWRKY33DCTD (AtW33DCTD), SIWRKY33A (SlW33A), SIWRKY33B (SlW33B) and AtWRKY25 (AtW25) driven by the CaMV 35S promoter. (A) Disease symptom development. Col-0 wild type, atwrky33 and transgenic atwrky33 lines constitutively expressing the various WRKY transgenes were sprayed inoculated with buffer (mock) or spores of Botrytis (Botrytis). The pictures of representative plants from two independent lines (indicated by numbers in red) for each transgene were taken at 4 dpi. (B) The expression of the Botrytis ActinA gene in spray-inoculated plants at 4 dpi. Total RNA of wild type, mutant and two independent lines of each transgene was isolated from leaf samples, and transcript levels were determined using qRT-PCR with Arabidopsis Actin2 gene as internal control. Error bars indicate SE (n=3). According to Duncan’s multiple range test (P=0.05), means of lesion areas do not differ significantly if they are indicated with the same letter.
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Figure 2: Complementation of atwrky33 mutant plants for Botrytis resistance by AtWRKY33 (AtW33), AtWRKY33DCTD (AtW33DCTD), SIWRKY33A (SlW33A), SIWRKY33B (SlW33B) and AtWRKY25 (AtW25) driven by the CaMV 35S promoter. (A) Disease symptom development. Col-0 wild type, atwrky33 and transgenic atwrky33 lines constitutively expressing the various WRKY transgenes were sprayed inoculated with buffer (mock) or spores of Botrytis (Botrytis). The pictures of representative plants from two independent lines (indicated by numbers in red) for each transgene were taken at 4 dpi. (B) The expression of the Botrytis ActinA gene in spray-inoculated plants at 4 dpi. Total RNA of wild type, mutant and two independent lines of each transgene was isolated from leaf samples, and transcript levels were determined using qRT-PCR with Arabidopsis Actin2 gene as internal control. Error bars indicate SE (n=3). According to Duncan’s multiple range test (P=0.05), means of lesion areas do not differ significantly if they are indicated with the same letter.

Mentions: For functional analysis of AtWRKY33 CTD, we determined whether its removal compromised the ability of AtWRKY33 to restore disease resistance and stress tolerance to the atwrky33 mutants. For this purpose, we generated a mutant AtWRKY33 protein in which the C-terminal 94 aa of AtWRKY33 was removed (AtWRKY33DCTD). Both AtWRKY33 and AtWRKY33DCTD gene were inserted into a plant expression vector behind the constitutive CaMV 35S promoter. For comparison, the coding sequence for AtWRKY25, which does not contain an extended CTD, was also placed into the same vector behind the strong promoter. All these three constructs were transformed into the atwrky33 mutant plants and transgenic plants constitutively expressing the transgenes were identified (Supplementary Fig. S1) and the progeny of two independent transgenic lines were tested for disease resistance to Botrytis based on both disease symptom development and accumulation of transcripts for Botrytis ActinA genes as an indicator for fungal growth on inoculated plants. As expected, the atwrky33 mutant was compromised in the disease resistance as indicated from enhanced disease symptoms after Botrytis infection (Fig. 2). The atwrky33 mutant plants were also compromised in heat tolerance at 45ºC as indicated from symptoms of mature plant development and reduced seedling survival rate after the heat shock (Fig. 3). Transformation of atwrky33 with the wild-type AtWRKY33 gene completely restored the Botrytis resistance and heat tolerance to the mutant (Figs 2, 3). Interestingly, in the transgenic atwrky33 mutant plants expressing AtWRKY25 or AtWRKY33DCTD driven by the strong CaMV 35S promoter, resistance to Botrytis was also largely restored as both disease symptoms and fungal growth were comparable to those in wild-type plants (Figs 2, 3). Both AtWRKY25 and AtWRKY33DCTD driven by the strong CaMV 35S promoter also restored the heat tolerance to the atwrky33 mutant plants when assayed using mature plants (Fig. 3A). The two genes also restored the heat tolerance to the atwrky33 mutant plants based on the increased survival rates of the transgenic plants, although growth of the surviving plants was significantly reduced during recovery following heat stress (Fig. 3B, C). Taken together, both AtWRKY25 and AtWRKY33DCTD genes driven by the strong CaMV 35S promoter largely restored both the Botrytis resistance and heat tolerance to the atwrky33 mutant plants.


Characterization of the promoter and extended C-terminal domain of Arabidopsis WRKY33 and functional analysis of tomato WRKY33 homologues in plant stress responses.

Zhou J, Wang J, Zheng Z, Fan B, Yu JQ, Chen Z - J. Exp. Bot. (2015)

Complementation of atwrky33 mutant plants for Botrytis resistance by AtWRKY33 (AtW33), AtWRKY33DCTD (AtW33DCTD), SIWRKY33A (SlW33A), SIWRKY33B (SlW33B) and AtWRKY25 (AtW25) driven by the CaMV 35S promoter. (A) Disease symptom development. Col-0 wild type, atwrky33 and transgenic atwrky33 lines constitutively expressing the various WRKY transgenes were sprayed inoculated with buffer (mock) or spores of Botrytis (Botrytis). The pictures of representative plants from two independent lines (indicated by numbers in red) for each transgene were taken at 4 dpi. (B) The expression of the Botrytis ActinA gene in spray-inoculated plants at 4 dpi. Total RNA of wild type, mutant and two independent lines of each transgene was isolated from leaf samples, and transcript levels were determined using qRT-PCR with Arabidopsis Actin2 gene as internal control. Error bars indicate SE (n=3). According to Duncan’s multiple range test (P=0.05), means of lesion areas do not differ significantly if they are indicated with the same letter.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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Figure 2: Complementation of atwrky33 mutant plants for Botrytis resistance by AtWRKY33 (AtW33), AtWRKY33DCTD (AtW33DCTD), SIWRKY33A (SlW33A), SIWRKY33B (SlW33B) and AtWRKY25 (AtW25) driven by the CaMV 35S promoter. (A) Disease symptom development. Col-0 wild type, atwrky33 and transgenic atwrky33 lines constitutively expressing the various WRKY transgenes were sprayed inoculated with buffer (mock) or spores of Botrytis (Botrytis). The pictures of representative plants from two independent lines (indicated by numbers in red) for each transgene were taken at 4 dpi. (B) The expression of the Botrytis ActinA gene in spray-inoculated plants at 4 dpi. Total RNA of wild type, mutant and two independent lines of each transgene was isolated from leaf samples, and transcript levels were determined using qRT-PCR with Arabidopsis Actin2 gene as internal control. Error bars indicate SE (n=3). According to Duncan’s multiple range test (P=0.05), means of lesion areas do not differ significantly if they are indicated with the same letter.
Mentions: For functional analysis of AtWRKY33 CTD, we determined whether its removal compromised the ability of AtWRKY33 to restore disease resistance and stress tolerance to the atwrky33 mutants. For this purpose, we generated a mutant AtWRKY33 protein in which the C-terminal 94 aa of AtWRKY33 was removed (AtWRKY33DCTD). Both AtWRKY33 and AtWRKY33DCTD gene were inserted into a plant expression vector behind the constitutive CaMV 35S promoter. For comparison, the coding sequence for AtWRKY25, which does not contain an extended CTD, was also placed into the same vector behind the strong promoter. All these three constructs were transformed into the atwrky33 mutant plants and transgenic plants constitutively expressing the transgenes were identified (Supplementary Fig. S1) and the progeny of two independent transgenic lines were tested for disease resistance to Botrytis based on both disease symptom development and accumulation of transcripts for Botrytis ActinA genes as an indicator for fungal growth on inoculated plants. As expected, the atwrky33 mutant was compromised in the disease resistance as indicated from enhanced disease symptoms after Botrytis infection (Fig. 2). The atwrky33 mutant plants were also compromised in heat tolerance at 45ºC as indicated from symptoms of mature plant development and reduced seedling survival rate after the heat shock (Fig. 3). Transformation of atwrky33 with the wild-type AtWRKY33 gene completely restored the Botrytis resistance and heat tolerance to the mutant (Figs 2, 3). Interestingly, in the transgenic atwrky33 mutant plants expressing AtWRKY25 or AtWRKY33DCTD driven by the strong CaMV 35S promoter, resistance to Botrytis was also largely restored as both disease symptoms and fungal growth were comparable to those in wild-type plants (Figs 2, 3). Both AtWRKY25 and AtWRKY33DCTD driven by the strong CaMV 35S promoter also restored the heat tolerance to the atwrky33 mutant plants when assayed using mature plants (Fig. 3A). The two genes also restored the heat tolerance to the atwrky33 mutant plants based on the increased survival rates of the transgenic plants, although growth of the surviving plants was significantly reduced during recovery following heat stress (Fig. 3B, C). Taken together, both AtWRKY25 and AtWRKY33DCTD genes driven by the strong CaMV 35S promoter largely restored both the Botrytis resistance and heat tolerance to the atwrky33 mutant plants.

Bottom Line: We compared AtWRKY33 with its close homologues to identify AtWRKY33-specific regulatory and structural elements, which were then functionally analysed through complementation.Thus, WRKY33 proteins are evolutionarily conserved with a critical role in broad plant stress responses.Both its CTD and promoter are critical for the uniquely important roles of WRKY33 in plant stress responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China Department of Botany and Plant Pathology, 915W. State Street, Purdue University, West Lafayette, IN 47907-2054, USA jie@zju.edu.cn zhixiang@purdue.edu.

No MeSH data available.


Related in: MedlinePlus